Tetracycline extractions



TETRACYCLINE EXTRACTIONS Pasquale P. Minieri, Brooklyn, N. Y., andWilliam M. Ziegler, Clementon, N. J., assignors to American CyanamidCompany, New York, N. Y., a corporation of Maine No Drawing. ApplicationMarch 28, 1955 Serial No. 497,454

2 Claims. (Cl. 260-559) This invention relates to improvements in thepurification of antibiotics. More particularly, this invention isconcerned with the extraction and purification of tetracycline fromfermentation broth impurities by formation of tetracycline-quaternaryammonium complexes, and the regeneration of the pure antibiotictherefrom.

In copending patent application, Serial No. 382,637, filed September 28,1953, now Patent No. 2,734,018, by Pasquale P. Minieri, Herman Sokol andMelvin C. Firman there is disclosed a process for recoveringtetracycline by precipitation of the antibiotic from its fermentationimpurities with quaternary ammonium salts such as alkyltrimethylammonium chloride and dialkyl dimethylammonium chloride. Theprecipitated complex is then extracted into an organic solvent such aschloroform or methyl alcohol and the purified antibiotic is regeneratedas the acid salt by the addition of a mineral acid. By increasing the pHrange of the acid salt solution to between about 3.0 and 7.0,isoelectric tetracycline base, as the trihydrate, is crystallized.

Tetracycline is a well-known broad spectrum antibiotic, capable ofexerting antimicrobial activity against a large number of microorganismsincluding both gram-positive and gram-negative species. As a member ofthe broad spectrum class of antibiotics, tetracycline possessesconsiderable advantages, these being of particular interest topracticing physicians. Clinically, it results in a lower incidence ofgastro-intestinal disturbances. Oral doses of this antibiotic cause arapid rise in therapeutic serum levels which are easily maintained bycontinued oral administration.

Because of the great demand for this antibiotic by the medicalprofession, the pharmaceutical industry is continuously searching forways and means to reduce manufacturing costs. Newly discoveredantibiotics, such as tetracycline, are usually obtained initiallythrough long and tedious fermentation and extraction processes which arenot only expensive but also time consuming. As more knowledge regardingthe chemistry and physical behavior of the antibiotic is accumulated,these long and laborious methods are-gradually shortened, resulting inthe elimination of many needless steps and the use of-expensive organicsolvents.

We have found that the extraction procedures disclosed in copendingapplication Serial No. 382,637, now Patent No. 2,734,018, can bemarkedly improved by eliminating the use of organic-solvents forextracting the tetracyclinequaternary ammonium complex. By completelyomitting the use of organic solvents to solubilize the complex, we haveovercome what has heretofore been accepted as a necessary requirement inthe quaternary-type purification process-of tetracycline. Our improvedprocessthus not only simplifies an unduly complex procedure, butcontrary to present belief, increases the-yields of tetracycline.

In accordance with'ourproces's, whole or "filtered tetra cycline broth,or an aqueous slurry of mesh solids is treated with a quaternaryammonium salt. The tetracycline-quaternary ammonium complex forms whenthe medium is at an alkaline pH range, but the quaternary salt may beadded either at an alkaline or at an acid pH. The complex is thenseparated and the tetracycline is extracted with an aqueous acidsolution at a pH of about 0.5 to about 3.0, preferably from about 1.0 toabout 2.5.- The acid salt of tetracycline which forms goes intosolution, from which it precipitates as a crystalline product uponincrease of the pH to the range from about 3.0 to about 7.0,precipitation starting at the lower value.

The order of addition of the quaternary ammonium compound and ofalkalinization is not critical. However, if a low potency broth is beingworked up, it is preferred that the quaternary ammonium compound headded first in order to insure maximum salt formation once the pH of themedium is increased. Thus, the quaternary ammonium compound may be addedto filtered broth at an acid pH followed by pH adjustment to thealkaline range or it may be added to whole broth at acid pH followed byincrease in pH to the alkaline range. Although the preferred pH rangefor complex formation is from about 8.0 to about 11.0, a range from pH6.5 to 11.5 is operable.

The usual techniques known to those skilled in the art may be employedto enhance the speed of the process or to increase the yield or both.Thus, as those familiar with antibiotic purification procedures wellknow, crystal formation is enhanced by seeding the acidic solution witha few crystals of tetracycline base. This induces more rapidcrystallization of the amphoteric substance. If the seeded solution isplaced in a refrigerator overnight instead of remaining at roomtemperature, a rich crop of pure tetracycline crystals forms which canbe readily separated by filtration and dried. Repeated seeding andrefrigeration increase the yield somewhat. Further refinements andmodifications may occur to those skilled in the art and it is intendedthat these be included Within the scope of the present invention.

It is preferable to separate the precipitated complex with the aid offilter-aid material inorder to ensure as complete a removal of theantibiotic as possible. We prefer to employ diatomaceousearth as thefilter-aid material in our process. However, it is not necessary toadhere strictly to this substance inasmuch as any inert, chemicallyinactive material may be employed as for example, cellulose powder,starch, paper pulp, silica gel, powdered glass, powdered rubber orsynthetic polymers. These materials are available on the open marketunder a variety of trademarked names.

The exact manner in which the tetracycline-quaternary ammonium complexformsis not presently known. However, inasmuch as the presence ofcertain polyvalent metallic ions such as calcium and magnesium isessential for complex formation, it is believed that the precipitate isorgano-metallic in nature, the linkage between the tetracycline and themetallic ions being unknown. Since the crude fermentation broth, thefiltered broth, or an aqueous slurry of mash solids may be employed asstarting materials in the process of this invention, the required ionsare inherently presentin such materials. If a non-metallic solution ormixture of tetracycline is used, then a small quantity of calcium ormagnesium ions maybe added thereto, either in the form of saltsor asfermentation impurities, in order to effect complex formation. These maybe added either before or after the admixture of the quaternary ammoniumsalt.

Any strong mineral acid may be employed for cleaving thetetracycline-quaternary ammonium complex as for example, hydrochloricacid, sulfuric acid, or phosphoric contains from 8 to 22 carbon atoms,and dialkyl dimethylanimonium chlorides wherein one of the two longchainalkyl groups contains from 1 to 22 carbon atoms and the remaininglong-chain alkyl group contains from 8 to 22 carbon atoms. This class ofquaternary ammonium salts may be represented by the general formula:

OHs HsC-ITI-Rl are available and which may be advantageously used in thepresent invention. Although the chlorides are the preferred andillustrated salts, the bromides, sulfates, 2, O

and nitrates can be used when available.

ARQUADS .--AVERAGE COMPOSITION 4 Still another group of quaternaryammonium compounds which have utility in our invention are thoseexemplified by the following general formula:

wherein R represents an alkyl or alkenyl radical containing from 7 to 21carbon atoms, inclusive, R represents a hydroxyalkyl radical containingfrom 2 to 4 carbon atoms, inclusive, and R represents an alkyl radicalcontaining from 1 to 4 carbon atoms, inclusive. As is well known, thesecompounds are generally referred to as iinidazoliniurn chlorides. Theyare available commercially under the trademark designation Quaternary O,Quaternary S, etc., the O'and S representing the length of the alkylchain attached to the ring, e. g, oleyl, stearyl, etc. The preferredquaternary compound in this series is Quaternary O which is l-methyl-1-(2-hydroxyethyl)-2-oleyl-imidazolinium chloride.

The invention is illustrated by the following examples which areintended merely to exemplify, not to limit, the scope of this invention.

1 Arquads 2G and 2Hl are diallryl dimcthylammonium chlorides. All othersare alkyl trimethylammonium chlorides.

wherein R is an alkyl chain containing from 8 to 18 carbon atomsinclusive or an esterified chain of the type:

wherein R' is an alkyl chain from 8 to 18 carbon atoms, inclusive, R" ishydrogen, a methyl or ethyl radical, R' is a methylene or dimethyleneradical attached by C-N linkage to the quaternary nitrogen atom, and Xand Y are interchangeably Example I Six liters of tetracycline wholebroth were acidified to pH 2.0 with sulfuric acid, and 75 ml. of Arquad16 added. The mixture was agitated 15 minutes, filtered withdiatomaceous earth, the cake washed, and the wash water added to thefiltrate. The combined filtrate and Wash was then adjusted to pH 8.8with ION sodium hydroxide, agitated 15 minutes, and filtered withdiatomaceous earth. The cake was washed with water, then slurried in 150ml. of water and acidified to pH 1.5 with sulfuric acid. This mixturewas agitated 15 minutes and filtered, the cake washed and the wash wateradded to the extract. The pooled extract and wash was adjusted to pH 3.8with ammonium hydroxide and seeded with crystalline isoelectrictetracycline. The resultant mixture was agaitated two hours and storedovernight in the refrigerator. The mixture was then filtered, the cakewashed with n-butanol and water, and air dried. There was obtained 35.3grams of tetracycline base, assaying 824 mcg.'/mg.

Example 11 Four liters of tetracycline whole broth were acidified to pH2.0 with sulfuric acid and agitated 15 minutes. 52 ml. of Arquad 16 wasadded, the mixture adjusted to pH 9.3 with 4 N sodium hydroxide,agitated 15 minutes and filtered with diatomaceous earth. The cake waswashed with water. One-half of the filter cake was slurried in 200 ml.of water and the mixture acidified to pH 1.5 with 4 N sulfuric acid. Themixture was agitated 15 minutes and filtered, the cake washed withwater, and the wash added to the extract. The combined wash and extractwas adjusted to pH 4.0 with ammonium hydroxide, seeded with crystallineisoelectric tetracycline, agitated three hours and filtered. The cakewas washed with nbutanol and water, an air-dried. There was obtained11.52 grams of tetracycline base, assaying 745 meg/mg.

Example 111 To 6 liters of whole tetracycline broth, at the harvest pH(6.0), were added 250 grams of diatomaceous earth. The mixture wasfiltered, the cake washed with water; then slurried in 5 liters ofwater. The mixture was acidified to pH 2.0 with sulfuric acid, agitated15 minutes, and

filtered. The cake was washed with water, and the wash in added to theextract. 70 ml. of Arquad 16 were added to the pooled extract and wash,the mixture was adjusted to pH 8.5 with 10 N sodium hydroxide, agitatedminutes and filtered. The cake was washed with water, then Example IV 60ml. of Arquad 16 was added to liters of tetracycline acid mash filtrate.The mixture was adjusted to pH 8.5 with sodium hydroxide, agitated 15minutes and filtered with 300 grams of diatomaceous earth. The cake waswashed with water, then slurried in 500 ml. of water and the mixtureacidified to pH 1.5 with sulfuric acid. The mixture was agitated 15minutes and filtered. The cake was washed with water, and the wash addedto the extract. pH 3.0 with ammonium hydroxide, seeded with crystallineisoelectric tetracycline, and further adjusted to pH 4.0 with ammoniumhydroxide. The mixture was agitated 3 hours and stored overnight in therefrigerator. The

crystalline slurry was then filtered, the cake washed and I air dried.There was obtained 15.46 grams of tetracycline base, assaying 888meg/mg.

The pooled wash and extract was adjusted to grams of Quaternary 0dissolved in 20 ml. ethanol. A filter aid, grams of diatomaceous earthwas added, the pH adjusted to 8.5 with sodium hydroxide and the mixturefiltered. The cake was suspended in 100 ml. of water adjusted to pH 1.5with sulfuric acid and filtered. The cake was washed with 25 ml. ofwater and the wash added to the filtrate. The pooled filtrates wereadjusted to pH 5.5 with ammonium hydroxide and stirred for 3 hours. Thesolids that formed were removed by filtration and air dried. There wasobtained 9.0 grams of tetracycline base, assaying 415 mcg./mg.

We claim:

1. The process of decomposing a quaternary ammomum-tetracycline complexselected from the group consisting of a long chainalkyltrimethyl-amm-onium chloridetetracycline complex and a long chaindialkyl-dimethylammonium chloride-tetracycline complex which comprisescontacting said complex With an aqueous acid solution at a pH within therange of about 0.5-3, separating the resulting aqueous phase containingthe tetracycline, and adjusting the pH of the aqueous phase with alkalito a pH within the range of about 37 so as to crystallize tetracyclinefree base therefrom.

2. The process according to claim 1 in which the quaternary ammoniumcompound is a mixture of long chain alkyltrimethylammonium chlorides andlong chain dialkyldimethylammonium chlorides in which the alkyl groupsconsist of hexadecyl, 6% octadecyl and 4% octadecenyl.

References Cited in the file of this patent UNITED STATES PATENTS2,516,080 Sobin et al. July 18, 1950 2,734,018 Minieri et a1. Feb. 7,1956 FOREIGN PATENTS 506,950 Belgium Nov. 30, 1951 OTHER REFERENCES VanDyck et al.: Antibiotics and Chemotherapy, vol. 2 (1952), pages 184 to196.

Bird et al.: Antibiotics and Chemotherapy, vol. 4, No. 7, July 1954,page 750.

1. THE PROCESS OF DECOMPOSING A QUATERNARY AMMONIUM-TETRACYCLINE COMPLEXSELECTED FROM THE GROUP CONSISTING OF A LONG CHAINALKYLTRIMETHYL-AMMONIUM CHLORIDETETRACYCLINE COMPLEX AND A LONG CHAINDIALKY-DIMETHYLAMMONIUM CHLORIDE-TETRACYCLINE COMPLEX WHICH COMPRISESCONTACTING SAID COMPLEX WITH AN AQUEOUS ACID SOLUTION AT A PH WITHIN THERANGE OF ABOUT 0.5-3, SEPARATING THE RESULTING AQUEOUS PHASE CONTAININGTHE TETRACYCLINE, AND ADJUSTING THE PH OF THE AQUEOUS PHASE WITH ALKALITO A PH WITHIN THE RANGE OF ABOUT 3-7 SO AS TO CRYSTALLIZE TETRACYCLINEFREE BASE THEREFROM.